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¹ SibTech, Inc., Newington, Connecticut; ² College of Pharmacy and ³ Department of Pathology, The Ohio State University, Columbus, Ohio; and ⁴ University of Connecticut, Health Center, Farmington, Connecticut

Requests for reprints: Marina V. Backer, Sibtech, Inc., 705 North Mountain Road, Newington, CT 06111. Phone: 860-953-1164; Fax: 860-953-1317. E-mail: mbacker{at}sibtech.com

Tumor neovasculature is a potential but, until very recently, unexplored target for boron neutron capture therapy (BNCT) of cancer. In the present report, we describe the construction of a vascular endothelial growth factor (VEGF)–containing bioconjugate that potentially could be used to target up-regulated VEGF receptors (VEGFR), which are overexpressed on tumor neovasculature. A fifth-generation polyamidoamine dendrimer containing 128 reactive amino groups was reacted with 105 to 110 decaborate molecules to produce a macromolecule with 1,050 to 1,100 boron atoms per dendrimer. This was conjugated to thiol groups of VEGF at a 4:1 molar ratio using the heterobifunctional reagent sulfo-LC-SPDP. In addition, the boronated dendrimer was tagged with a near-IR Cy5 dye to allow for near-IR fluorescent imaging of the bioconjugate in vitro and in vivo. As would be predicted, the resulting VEGF-BD/Cy5 bioconjugate was not cytotoxic to HEK293 cells engineered to express 2.5 x 10⁶ VEGFR-2 per cell. Furthermore, it showed binding and activation of VEGFR-2 comparable with that of native VEGF. Internalization of VEGF-BD/Cy5 by PAE cells expressing 2.5 x 10⁵ VEGFR-2 per cell was inhibited by excess VEGF, indicating a VEGFR-2-mediated mechanism of uptake. Near-IR fluorescent imaging of 4T1 mouse breast carcinoma revealed selective accumulation of VEGF-BD/Cy5, but not BD/Cy5, particularly at the tumor periphery where angiogenesis was most active. Accumulation of VEGF-BD/Cy5 in 4T1 breast carcinoma was diminished in mice pretreated with a toxin-VEGF fusion protein that selectively killed VEGFR-2-overexpressing endothelial cells. Our data lay the groundwork for future studies using the VEGF-BD/Cy5 bioconjugate as a targeting agent for BNCT of tumor neovasculature.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁵ M. V. Backer, unpublished.

⁶ M. V. Backer, unpublished.

Received 5/18/05; revised 6/30/05; accepted 7/15/05.